Sample support

ABSTRACT

The sample support comprises at least one sample receiving chamber for a sample liquid, and a distributor channel for sample liquid connected to said at least one sample receiving chamber, with at least one such distributor channel extending from each sample receiving chamber. The sample support further comprises at least one reaction chamber entered by an inflow channel branched off said at least one distributor channel, and a venting opening for each reaction chamber. Each distributor channel and each inflow channel are dimensioned to have the liquid transport through the distributor and inflow channels effected by capillary forces. In each reaction chamber, the entrance region of the inflow channel is provided with a means for generating a capillary force causing the sample liquid to flow from the inflow channel into the reaction chamber.

CROSS REFERENCES

This application is a divisional of, and claims priority to, applicationSer. No. 09/623,910. Ser. No. 09/623,910 is the U.S. national phase ofinternational application No. PCT/EP99/01607, filed on Mar. 11, 1999.Foreign priority is claimed to German patent application No. 198 10499.5, filed on Mar. 11, 1998, and to German Patent application No. 19902 309.3, filed on Jan. 21, 1999. The contents of each of thoseapplications are incorporated by reference in the present application.

The invention relates to a sample support of the type used formicrobiological examinations performed on sample liquids as well as formedical and environmental analysis and diagnostics.

In microbiological diagnostics, use is made of optical methods such asabsorption, scattering and luminescence analyses, e.g. for transmission,fluorescence or turbidity measurements. Such processes are carried outusing sample supports or test strips made of transparent plastic andcomprising a plurality of chambers or cup-shaped deepened portionsformed with one open side. The sample supports or test strips comprisee.g. 32 or 96 chambers or deepened portions having a reagent arrangedtherein. After inoculation with a bacterial suspension, the samplesupports or test strips are sealed by a transparent film or closed by alid, if required. The deepened portions have a filling volume from 60 μlto 300 μl and are filled individually by means of auxiliary apparatus;pipettes having one channel or 8, 48 or 96 channels are used for thispurpose.

From U.S. Pat. No. 4,038,151, a sample plate for an automated opticalexamination method is known, serving for the detection and counting ofsuspended micro-organisms and for determining their sensitivity toantibiotics. The plate is made of rigid transparent plastic andcomprises e.g. 20 conic reaction chambers. The cross-sectional area ofthe reaction chambers is larger on one side of the plate than on theother side. Provided next to each reaction chamber are two overflowchambers which are located on that side of each reaction chamber wherean inflow channel for the respective reaction chamber is arranged.

The reaction chambers are connected to overflow chambers via slits. Thereaction chambers, the slits and the overflow chambers extend over thecomplete thickness of the sample plate. The reaction chambers areconnected in groups, via specially arranged and shaped inflow channelsarranged on one plate side, to at least one sample receiving chamberclosed by a septum. The inflow channels tangentially open out on thelarger side of the conical reaction chamber. The form and the surface ofthe cross section of each inflow channel are formed with an abruptchange at a respective site. On these sides—when viewed in the flowdirection—a flat and wide channel undergoes a transition into a deep andsmall channel. The inflow channels arranged on one plate side may belonger than the respective shortest connection between the reactionchamber and the sample receiving chamber so that a back diffusion ofcomponents arranged in the suspension will be rendered more difficult.The plate—except for an edge region—is on both sides bonded to arespective semipermeable film covering the reaction chambers, theoverflow chambers, the slits, the inflow channels arranged on one sideof the plate, as well as one side of the sample receiving chamber. Thereaction chambers are covered by a dried layer of a reagent substance.

For introducing the sample liquid into the known sample plate, thechannels and chambers of the sample plate are evacuated so that thesample liquid is passed from a container arranged externally of theplate via a cannula through the septum from the edge of the plate intothe sample receiving chamber, and will flow via the inflow channels intothe reaction chambers and, if required, into the overflow chambers. Thesuspension (sample liquid) flown into the reaction chamber and thereagent layer are in contact with the adhesive layer arranged on thefilm.

During the optical examination of the samples in the reaction chambers,the sample plate is arranged vertically in the measuring device. In thisorientation, the inflow channels, relative to the direction of gravity,are arranged to enter the reaction chamber from above, and the overflowchambers lie above the reaction chambers. Thus, gas bubbles whichpossibly exist in the reaction chamber or are generated in case of areaction or a metabolic process, can accumulate in the overflow chamberswithout disturbing the optical examination of the samples.

From U.S. Pat. No. 5,670,375, a sample plate is known whose cavities,provided in a number of up to 64, are inoculated simultaneously. Afterthe air has been sucked from the cavities, the fluid under examinationwill flow from a container arranged externally of the sample plate via aconnecting tube into the cavities and thus will fill the latter.

Known from U.S. Pat. No. 5,223,219 is a sample support wherein, startingfrom a sample infeed region, sample liquid enters the reaction chambersvia a distributor channel system. The reaction chambers contain porousinserts provided with reagents. By the capillary forces generated in theporous inserts, the sample liquid is “sucked” into the reactionchambers. The fact that the reaction chambers have inserts arrangedtherein, imposes restrictions on the photometric examinations of thesample liquids arranged in the reaction chambers and reacting with thereagents. Thus, for instance, this arrangement does not offer thepossibility to perform transmitted-light measurements andoptical-turbidity measurements.

Finally, the state of the art also includes liquid distributor systemsfor transporting a sample liquid from an ampoule into a plurality ofreaction chambers wherein, in these systems, the force of gravity isutilized for generating a liquid flow through the distributor channels.The reaction chambers have to be vented, which is performed by ventingchannels originating from the reaction chambers and by themselvesforming a system of venting channels. Both of these channel systems(distributor channel system and venting channel system) are designed inthe manner of communicating tubes, which—since gravity isutilized—prevents that the sample liquid might leak from the ventingchannels after the reaction chambers have been filled.

The increasing widening and automation of quasi-parallel examinations ofmicrobiology and of analytical and diagnostic procedures require thatthe existing sample-support and sample-liquid distributor systems befurther developed and particularly be miniaturized. Due to the thusresulting relatively small cross-sectional areas of the channels, it isdesirable to use other forces than gravity and pressures for liquidtransport. In this regard, particularly capillary forces would appearuseful, which, however, would make it difficult to maintain the liquidtransport even when the liquid is to flow from a region of a smallercross section into a region of a larger cross section within the samplesupport and the sample liquid distributor system, respectively.

Thus, it is the object of the invention to provide a sample support anda sample liquid distributor system which have a relatively high densityof reaction chambers per unit area, which can be produced at low costsand which include a liquid flow control mechanism controllable in asimple manner from outside.

According to the invention, the above object is achieved by providing asample support and a sample liquid distributor system, respectively,comprising

-   -   at least one sample receiving chamber for a sample liquid,    -   a distributor channel for sample liquid, connected to said at        least one sample receiving chamber, with at least one such        distributor channel extending from each sample receiving        chamber,    -   at least one reaction chamber entered by an inflow channel        branched off said at least one distributor channel, and    -   a venting opening for each reaction chamber.

This inventive sample support and this inventive sample liquiddistributor system is characterized in

-   -   that each distributor channel and each inflow channel are        dimensioned to have the liquid transport through the distributor        and inflow channels effected by capillary forces, and    -   that, in each reaction chamber, the entrance region of the        inflow channel is provided with a means for generating a        capillary force causing the sample liquid to flow from the        inflow channel into the reaction chamber.

According to the invention, it is provided that the distributor channelsand inflow channels have cross sectional areas of such small size andcross sectional areas of such shapes, respectively, that the liquidtransport therein is performed by capillary forces. Thus, the channelsare formed as capillaries. The reaction chambers provided to receive thesample liquid flowing via the channels, have a larger cross section thanthe inflow channels. In this manner, a situation is created where theliquid has to flow from a channel of a smaller cross section into alarger cavity, i.e. a reaction chamber. To have this flow performedexclusively under the effect of capillary forces, it is providedaccording to the invention that, in each reaction chamber, notably inthe entrance region of the inflow channel, structures formed on theinner side of the reaction chamber or asymmetries are provided as meansfor generating a capillary force enabling a flow of the sample liquidfrom the inflow channel into the reaction chamber. By the provision ofsuch capillary-force generating means in the entrance region of aninflow channel into a reaction chamber, the sample liquid flow generatedby capillary forces is maintained until the reaction chamber has beenfilled. These capillary-force generating means enhance the wetting ofthe walls of the reaction chamber with sample liquid, thus maintainingthe liquid flow constant. By way of alternative to the above mentioneddesigns of the capillary-force generating means, these can also beprovided by surface treatment of the reaction chambers to the effectthat these surfaces are made hydrophilic, or are made hydrophilic tosuch an extent that the interior sides of the reaction chambers arewetted and the reaction chambers are completely filled with sampleliquid.

Particularly, the capillary-force generating means in the entranceregion of the inflow channels into the reaction chambers are realized bythe provision of an inflow groove or the like. This inflow groovecomprises at least two limiting faces connected to each other by atransition region. This transition region is provided with roundedregions whose radii are small enough to generate the capillary forcesrequired for the flow of the sample liquid along this groove. If theinflow channel is arranged to discharge into the reaction chamber at theheight of the bottom face, then, by suitable selection of the roundingradius in the region between the bottom face and the side faces of thereaction chamber, the liquid flow can be maintained in that the liquidwill first flow along the corner and transition regions between thebottom face and the side faces to thus wet the whole bottom area,whereas, from that point, the further transport will be maintained bythe capillary effect of the reaction chamber whose cross section is nowcompletely filled with sample liquid. In a case where the inflow channelis arranged to enter the reaction chamber from above the bottom face outof one of the side faces of the reaction chamber, a groove or a similarfurrow-like deepening should be formed in the respective side wallbetween the entrance and the bottom face. Such a groove can alsosuitably be provided by the corner region of two side faces of thereaction chamber extending to each other at an angle, provided that therounding radius in the corner or transition region of both side faces issmall enough to generate capillary forces acting on the sample liquidwhich are large enough to “pull” the sample liquid from the inflowchannel. As to the required radii of curvature of these grooves, itshould be generally observed that these are made smaller than thesmallest dimension of the channel joined by the grooves.

By way of alternative to the capillary-force generating means, it can beprovided that the channels extend under an angle other than 90° out froma face delimiting the chamber. Due to the resultant non-circularentrance opening, the sample liquid will in the most favorable case flowfrom the channel in to the chamber without additional measures.

The mechanism causing the sample liquid under examination to flow fromthe sample receiving chambers into the distributor channels, canlikewise be obtained by use of structures generating capillary forces.In the simplest case, the distributor channels are arranged to branchoff from the sample receiving chambers at the height of the bottom facesof the chambers. Since, after the filling of the sample receivingchambers with sample liquid, the cross section of the distributorchannels are wetted with liquid in the entrance region, a flow withinthe distributor channels will be generated automatically. The dischargeof the sample liquid from the sample receiving chambers is thusguaranteed.

A different situation exists if, usually for reasons of productiontechnology, the distributor channels are arranged to enter the samplereceiving chambers from above the bottom faces. In this case, it must beprovided that the sample liquid is “pulled upward” starting from theliquid level within the sample chambers. This is effected by acapillary-force generating means, arranged in the sample receivingchamber, which can be configured in the same manner as thecapillary-force generating means arranged in the reaction chambers. Alsoin this case, a preferred variant comprises a groove formed as anoutflow groove in one of the side walls of the sample receivingchambers. As an alternative thereto, the groove can be provided as atransition region and corner region between two mutually angles sidefaces of the sample receiving chambers. In all of such cases, care mustbe taken that, by selecting a correspondingly small rounding radius ofthe groove and corner region, respectively, capillary forces aregenerated to cause the liquid to flow automatically.

As evident from the above description, miniaturization offers thepossibility to arrange a large number of reaction chamber within anextremely small space, with the reaction chambers provided e.g. ascavities formed in a base body. As to the distribution of the sampleliquid via the distributor channels and the inflow channels branchingoff therefrom, it is desirable that the sample liquid be caused to fillall of the reaction chambers in the most uniform manner possible andparticularly simultaneously. To guarantee this effect—or largelyguarantee it—in the distributor channel system provided according to theinvention, the inflow channels should suitably have a smaller crosssectional area than the distributor channels. Thus, the inflow channelswill act in the manner of throttles decelerating the liquid transportwhich is still generated by capillary forces. All of the inflow channelsbranching off along the length of the distributor channel can have thesame cross sectional areas. Alternatively, the cross sectional areas ofthe inflow channels can be widened with increasing distance of theinflow channels from the sample receiving chamber, so that, in thoseinflow channels which branch off first—relative to the flow direction ofthe sample liquid through the distributor channels—a larger throttleeffect is obtained than in the inflow channels branching off later.

For reasons of space, the inflow channels are suitably arranged tobranch off from the distributor channels on both sides thereof. In thisregard, under the aspect of flow technology, two branch-off sites of thedistributor channel which have mutually opposite inflow channelsbranching off therefrom on opposite sides, should advantageously not bearranged directly opposite each other but at a mutual displacement alongthe length of the distributor channel. Notably, each inflow channelbranching off from the distributor channel will disturb, although justslightly so, the liquid transport maintained by the capillary forces.For these reasons, such disturbances should not at the same time affectthe liquid front moving along the distributor channels, which would bethe case if two mutually opposite, branched-off inflow channels were tobranch off at the same height of the distributor channel and/or directlyopposite each other.

To make it possible that sample liquid can flow into the reactionchambers from the sample receiving chambers, it must be provided thatthe gas contained in these chambers and in the channel system leadingthereto is allowed to escape. For this reason, each reaction chamber isprovided with a venting opening. If these venting openings are wetted oreven covered while the reaction chambers are being filled with sampleliquid, a danger exists that the sample liquid escapes from the reactionchambers via the discharge openings if the wetting and covering of theventing openings can cause large enough capillary forces therein. Infact, it is desirable that the reaction chambers be completely filledwith sample liquid because any gas which might still have entered wouldmake the optical examination by photometry more difficult or evenimpossible.

Advantageously, further transport of the sample liquid through theventing openings is prohibited by use of means preventing further flowof sample liquid. Such means are advantageously based on the principleof utilizing geometric shapes of the venting openings and of possiblyjoining venting channels to make the generated capillary forces smallenough to cause an interruption of the sample liquid flow. To beparticularly preferred in this regard are so-called “capillary jumps”,i.e. enlargements of the channels into which the sample liquid cannotflow by because of more-difficult wetting conditions on the walls of thewidened channel portions. For instance, venting channels joining theventing openings can be arranged to enter a cavity and a widened portionof the channel, wherein the entrance region is arranged within a sidesurface of the widened channel portion or cavity and no or few cornerregions are arranged around the entrance region. This is providedbecause each corner region would again generate capillary forces whichin turn are determined by the extent of the rounding.

Suitably, the venting openings of the reaction chambers are followed byconnection channels entering a venting collecting channel. This ventingcollecting channel is provided with a venting opening which connects theventing system of the sample support with the environment. Since thereis thus provided a second distributor channel system which from acentral site, i.e. the venting collecting channels, allows for a fluidconnection to the individual reaction chambers, it is desirable toutilize this second distributor system for a well-aimed introducing ofadditional reagent liquids into the reaction chambers. By introducingadditional reagent liquids, the sample liquids which in the reagentchambers have already undergone a reaction with a reagent substance thathad been introduced thereinto in advance and arranged therein e.g. indried form, can be subjected to a second reaction. Since, however, theventing system is already provided with a means, particularly in theform of widened channel portions, which is to prevent a liquid flow fromthe reaction chambers via the venting openings, such means will alsoimpede the transport of the reactive liquid via the venting channelsystem into the reaction chambers. In this regard, it is of advantageif, by a corresponding configuration of the widened channel portionsforming the flow prevention means, it is safeguarded that the flow ofreagent liquid into the widened channel portions under the effect ofcapillary forces is taking place. In this regard, use can be made againof the inflow groove structures described already further above whichcan be realized by correspondingly designed corner regions in thetransition region of a plurality of mutually angled faces of the widenedchannel portions.

By providing the widened channel portions with capillary forcegenerating means allowing the inflow of reagent liquid into the widenedchannel portions, the latter are filled with reagent liquid until thereagent liquid covers the entrance region of the portions of the ventingchannels from the reaction chambers. Thus, in this entrance regions, thetwo reagent liquid and sample liquid fronts will contact each other. Thefurther transport of the reagents will now be performed by diffusion upinto the reaction chambers.

The well-aimed filling of the widened channel portions for effecting thediffusion transport of the reagents, can alternatively be obtained alsoby introducing a control liquid (which is inert toward the reagents andthe sample liquids). For this purpose, a control channel is arranged toenter the widened channel portion, with the control liquid reaching thewidened channel portion via this control channel. In this manner, aliquid-controlling valve is provided, which, as it were, allows for asingle actuation for switching the valve from the closed condition intothe open condition with regard to the possibility of a diffusiontransport of the reagents. The introducing of the control liquid intothe widened channel portions can be carried out by application ofpressure or again by use of capillary forces. For this purpose, use canbe made again of the same mechanisms and designs of the side walls andentrance regions that have been described further above.

The introducing of the reagent liquid into the venting collectingchannel and the venting channel system, respectively, of the reactionchambers is suitably performed in that this channel system is in fluidconnection with at least one reagent liquid receiving chamber. From thischamber, the reactive liquid will be discharged particularly by use ofthose mechanisms described further above in connection with the samplereceiving chambers and the distributor channels.

For the examination of microbiological samples using the inventivesample support, it may be required that the sample under examination beamplified beforehand, i.e. that the quantity of the sample material beincreased before the material is fed to the individual reaction chambersvia the distributor inflow channel system. The process of the amplifyingand of the introducing the amplified sample into sample receivingchambers is simplified if the amplification itself is performed at thesite of the sample receiving chamber. In this case, it is desirable thatthe amplified sample material is supplied, under external control, tothe reaction chambers assigned to the sample receiving chambers.According to an advantageous variant of the invention, this is performedin that, between the sample receiving chamber and the first inflowchannel branching off the at least one connecting channel, a first valveis arranged which is preferably arranged as a one-way valve which can beswitched from its closed condition into its open condition only once. Ifthe transport of the sample from the sample receiving chamber to theindividual reaction chambers is performed by capillary forces—which isto be preferred, and which is why all of the channels in the samplesupport are formed as capillaries—then this first valve can also bearranged in the venting channel which is associated with a group ofreaction chambers connected to the sample receiving chamber. Notably, bythe thus obtained controlled venting of the reaction chambers, theinflow of the sample material from the sample receiving chamber to theindividual reaction chambers will be controlled.

The “interface” of the inventive sample support for driving the firstvalve or the first valves should be of the simplest possibleconfiguration. This necessitates that the valve can be controlled in asimple manner from an external site. Preferably, it is provided that thevalve be controlled hydraulically or pneumatically, notably by theliquid and respectively the gas on this valve. Particularly, forinstance, by applying a pressure pulse on the sample material containedin the sample receiving chamber, a hydraulic pressure is generated onthe first valve which will overcome or otherwise bridge the lockingelement of the first valve. Thus, for instance, it is possible to designthe first valve as a burst valve comprising a burst film designed toburst open when a specific pressure is exceeded, thus opening thechannel in which the valve is arranged. By way of alternative, flapvalves or back-check valves can be used which will open when acorresponding pressure of the applied fluid (liquid or gas) is reached.This type of valves is preferable particularly if the transport of thefluids through the sample support is performed by application ofpressure, i.e. not through capillary forces.

A further alternative of the design of the first valve or the firstvalves resides in that this valve is of a hydrophobic design which isrealized by a corresponding surface treatment of the channel in theregion of the valve or by an insert portion. The fluid applied to thehydrophobic valve will bridge the valve e.g. as a result ofa—particularly pulse-like—application of pressure. When the channel inthe region of the valves is in this manner wetted with liquid and use ismade of capillary forces for the further transport of the liquid, theseprovisions will generate a one-way valve which can be externally bridgedin a simple manner, i.e. by applying pressure onto the sample receivingchamber.

Further, the first valve can advantageously provided as a widenedportion of the channel, which in turn will act as a capillary jump. (Inthis regard, cf. the description in connection with the venting channelsfurther above.) As soon as this widened channel portion has been filledwith liquid, which is performed e.g. by corresponding application ofpressure to the sample receiving chamber or externally by introducing aseparate or control liquid, the transport of the liquid behind thevalve, caused by capillary forces, will be safeguarded so that the valveitself can be bridged again hydraulically.

All of the channels, chambers and the like structures are placed,preferably from one side, in a base body covered in a liquid-tightmanner by a lid body, particularly a film. Alternatively, both bodies,the base body and the lid body, can together form the channels andcavities. The sample support is preferably made of plastic, such aspolystyrene or polymethyleneacrylate (PMMA), polycarbonate or ABS. Thesample support can be produced by casting respectively one shaped insertin a micro-injection mold. In this case, the structure of the shapedinsert is complementary to the structure of the base body and/or the lidbody. The shaped inserts to be used for these injection moldingtechniques are produced by lithography or galvanoplasty, by microerosionor by micromechanic treatment such as diamond machining. Further, thestructured elements of the sample support can be produced from aphoto-etchable glass or from silicon by anisotropic etching or bymicromechanic treatment processes. The components of the sample support(base body and lid body) are connected to each other on their contactingfaces, particularly by ultrasonic welding. In any case, this connectionmust be liquid- and gas-tight so that the individual chambers andchannels will not be in mutual contact via contacting faces of theelements from which the sample support (base body and lid body) is made.

The inventive sample support can comprise transparent material for usein transmitted-light measurements, and transparent or non-transparentmaterial for luminescence measurements. If the sample support is madefrom several components (base body and lid body), the individualcomponents of the sample support can comprise different materials.

The height of the reaction chambers and thus the thickness of the liquidlayer having the light passing therethrough can be adapted to theoptical evaluation method. Within the sample support, reaction chamberswith different heights can be arranged.

The inventive sample support can comprise reaction chambers with volumesin the range from 0.01 μl to 10 μl. The density of the reaction chamberscan be up to 35/cm². Thus, one sample support of a handy size can easilyaccommodate 50 to 10,000 reaction chambers. The individual channels havea width and depth of 10 μm to 1,000 μm and particularly 10 μm to 500 μm.

A sample support configured according to the invention has a height ofe.g. 4 mm, wherein, for a two-part configuration (base body and lidbody), the base body has a thickness of about 3.5 m and the lid body,provided as a film, has be thickness of 0.5 mm. The reaction chambers,which—if desired—are round but may also be edgy, have a depth of about3.0 mm so that the bottom wall will have a thickness of 0.5 mm. Thevolume of these reaction chambers is respectively 1.5 μl. The individualchannels particularly have a rectangular cross section, wherein theinflow channels have a width of about 400 μm and a depth of 380 μm, andthe distributor channels having the inflow channels branching offtherefrom have a width of about 500 μm and a depth of about 380 μm. Theventing openings (in case of a rectangular cross section) are about 420μm wide and about 380 μm deep. The venting channels joining the ventingopenings particularly have a width and depth of 500 μm and 1,000 μm,respectively. A surface of 21.5 mm×25 mm, i.e. of 540 mm², has arrangedthereon 96 reaction chambers suited to be filled simultaneously. Thus,under the arithmetic aspect, the area required by the reaction chamberis 5.6 mm².

The inventive sample support particularly has the following advantages:

-   -   The sample support contains a substantially larger number of        reaction chambers with smaller volumes, resulting in a larger        density of the sample cambers.    -   Filling the reaction chambers with the sample liquid is        performed faster and—while requiring lesser apparatus        components—in a simpler manner, since the sample liquid will be        applied only at a few sites (sample receiving chambers) and will        automatically flow from there into the reaction chambers under        the effect of capillary forces.    -   Filling the reaction chambers requires neither an overpressure        of the sample liquid nor an underpressure in the reaction        chambers.    -   The sample receiving chambers are filled by use of devices of        commercially available types, with the sizes and volumes of the        sample receiving chambers being adapted to such devices.    -   In a sample support provided with sample receiving chambers for        the reagent liquid, a reagent liquid existing in a liquid can be        easily introduced at a later time into the reaction chambers        already filled with a fluid.    -   The sample material can be introduced in a well-aimed manner        from the sample receiving chamber into the individual reaction        chambers, notably by provision of a first valve in the channel        system completely joining the sample receiving chamber.    -   Also the reagent liquid, which—if desired—is fed into the        reaction chambers from their venting side, can be introduced        into the reaction chambers in a controlled manner due to the        provision of second valves in the venting duct. These second        valves can be controlled particularly hydraulically,        pneumatically and in similar manners, as is the case for the        first valves.    -   The covered reaction chambers are completely filled with the        fluid under examination. The filing volume of each reaction        chambers is determined automatically; a dosage means for each        individual reaction chamber is not required.    -   During a possible further treatment and during measurement, the        fluid contained in the reaction chambers is effectively        protected from evaporation by the cover film tightly connected        to the base body.    -   The material required for introducing a reagent into the        reaction chambers, the required testing material, e.g. blood        suspension, blood samples or active substances, and thus the        costs, are less than in sample supports with reaction chambers        having larger volumes.    -   For the fluid under examination, e.g. a bacterial suspension,        sample receiving chambers can be provided which are arranged in        the base body or in the lid body and which, if desired, have a        plurality of connecting channels entering thereinto.    -   The microbiological, microchemical or bacteriological        examination of the samples introduced into the sample support        can be fully automated while the expenditure for the measuring        devices is reduced.    -   The sample support can be stored at normal room temperature. The        space requirement for storage is distinctly less than in        conventional sample supports.    -   The sample supports, in analogy with known sample supports, are        designed for single use. Because of the enlarged packing density        of the reaction chambers, the volume of used sample supports to        be disposed of is smaller than when using conventional sample        supports.

By use of an adapted miniaturized device, the reaction chambers in thesample support can be provided with a chemically or biologically activereagent which after the introducing of the reagent fluid will be driedand adhere on the bottom and the wall of the reaction chambers. Usefulas reagents are e.g. oligopeptide-β-NA-derivates,p-nitrophenyle-derivates, sugar for fermentation examinations and otherexaminations, organic acids, amino-acids for assimilation examinations,decarboxylase substrates, antibiotics, antimycotics, nutrientsubstrates, marker substances, indicator substances and othersubstances.

The inventive sample support which to be provided with a reagent, ifrequired, can be used for the biochemical detection and the sensitivitytesting for clinically relevant microorganisms. In a fully automated andminiaturized system. there is produced a defined suspension ofmicroorganisms which is delivered to the sample support. The inoculatedsample support is—possibly after a further treatment—measured by use ofan optical method. The results obtained thereby are picked up under theassistance of a computer and are mathematically examined and evaluatedthrough suitably adapted methods.

The inventive sample support is useful in blood-group serology, inclinical chemistry, in the microbiological detection of microorganisms,in testing the sensitivity of microorganisms to antibiotics, inmicroanalysis and in the testing of production materials.

The invention will be explained in greater detail with reference to theFigures.

FIG. 1 is a plan view of the upper side of a sample support, with thecover film partially broken away,

FIG. 2 is a sectional view, taken along the line II-II in FIG. 1, of asample receiving chamber with a distributor channel joining the same,

FIG. 3 is a sectional view, taken along the line III-III, of the samplechambers, showing also the distributor channels branching off therefrom,

FIG. 4 is a sectional view, taken along the line IV-IV in FIG. 1, of thereaction chambers arranged adjacent each other along the width of thesample support,

FIG. 5 is a view of the area of the sample support marked by V in FIG.1, in perspective view and enlarged representation,

FIGS. 6 to 9 are cross-sectional views, taken along the lines VI-VIthrough IX-IX in FIG. 5, illustrative of the configuration of thechannels and chambers respectively in their transition regions andentrance regions, and

FIGS. 10 to 14 are views of different valve configurations in plan andsectional views, with the valves arranged in the region marked by XI inFIG. 5.

The sample support 10 illustrated in the drawing is of a two-partstructure and comprises a base plate 12 whose upper side 14, shown inFIG. 1, is covered by a cover film 16 (cf. also FIGS. 2 to 4). Samplesupport 10 is provided to direct applied sample liquid into a pluralityof reaction chambers under the effect of gravity, with the reactionchambers having different reagent substances arranged therein. Further,it is required that the reaction chambers filled with sample liquid canbe photometrically examined. Further, it is provided that liquid can beinserted into the reaction chambers in a controlled manner fromdifferent sites.

As particularly evident from FIG. 1, sample support 10 is divided into aplurality of sections 18 of mutually identical configurations. In thesubsequent description, reference is made each time to the configurationof one such section. Within each section 18, the base plate 12 of samplesupport 10 is provided with a structured surface on its upper side 14,which is realized by forming grooves and deepened portions into the baseplate 12 from upper side 14. All of the grooves and deepened portionsconstitute a sample-liquid and reagent-liquid distributor system whichtowards the upper side of sample support 10 is covered by cover film 16.

Each section 18 of sample support 10 includes a sample receiving chamber20 for receiving a sample liquid 22 (cf. FIG. 2). Arranged in fluidconnection with the sample receiving chamber 20 is a distributor channel24 entering the sample receiving chamber 20 on the upper end of thechamber. Inflow channels 26 extend from distributor channel 24 on bothsides thereof and in a serpentine configuration when seen in plan viewaccording to FIG. 1, which channels like the distributor channel 24 aregenerated by the formation of grooves in the upper side 14 of base plate12. The inflow channels 26 extend from distributor channel 24 to thereaction chambers 28 which are arranged as deepened portion formed inbase plate 12 from upper side 14. Connecting (venting) channels 30extend from the reaction chambers 28. These connecting channels 30 arearranged to enter group-wise into two venting collecting channels 32extending in parallel to each other and in parallel to the distributorchannel 24. In other words, the reaction chambers 28 arranged on bothsides of distributor channel 24 extend between distributor channel 24 onthe one hand and one of the two venting collecting channels 32 on theother hand. Also the connecting channels 30 and the venting collectingchannels 32 are generated by the formation of grooves in the upper side14 of base plate 12. Further, the venting collecting channels 32 havetheir upper ends terminating in a venting opening 34 formed in an outeredge side 36 (cf. FIG. 2) of base plate 12. The respective end of theventing collecting channels 32 which is arranged opposite these ventingopenings 34, is connected to a reagent liquid receiving chamber 38 to bediscussed later. Also this chamber 38 is realized by forming a deepenedportion in the upper side 14 of base plate 12.

The transport of sample liquid 22 from a sample receiving chamber 20 ofa section 18 of sample support 10 into the reaction chambers 28 assignedto sample receiving chamber 20 is performed by use of capillary forces.The same applies to the transport of reagent liquid from chambers 38into reaction chambers 28. To make it possible that these capillaryforces are generated within the channels, these channels 24,26,30,32have to be dimensioned in a suitable manner. If required, the innersides of the channels have to be subjected to a surface treatment torender these surfaces hydrophilic. Whether such a treatment is required,will depend on the material of base plate 12 and cover film 16 on theone hand, and on the viscosity and the nature of the to-be-transportedliquids (sample liquid and reagent liquid) on the other hand.

While the utilization of the capillary forces within the channels can berealized in a simple manner by the above described measures, achieving areliable transport of liquid from the chambers 20,38,28 into theconnected channels and respectively out from the channels 26 into theconnected reaction chambers 28, is problematic. With regard to the fluidconnection of distributor channel 24 to the sample receiving chamber 20,a problem resides particularly in that the entrance site 40 ofdistributor channel 24 into sample receiving chamber 20 is located abovethe bottom wall 42 of chamber 20 and within the lateral delimitation 44of chamber 20. The lateral delimitation 44 of chamber 20 is formed byside face portions 46 As can be seen particularly in FIG. 1, the sidefaces 46 extend in angular orientations in the region below the entrancesite 40, in this case under a mutual angle of about 90°, so that acorner region 48 is generated between both side faces 46. This cornerregion 48 has such a small radius of curvature on its bottom that thereis formed an outflow groove 50 in which a liquid meniscus is generatedupon wetting with sample liquid 22. In the instant case, this outflowgroove 50 extends transverse to bottom wall 42. Thus, as a result of thewetting of the side faces 46 in the corner region 48, capillary forcesare generated in the outflow groove 50, which forces are sufficient toact on the sample liquid 20 to the effect that the sample liquid 22 issucked from sample receiving chamber 20 into distributor channel 24. Theoutflow groove 50 extends particularly all the way to the bottom wall 42of sample receiving chamber 20. As soon as the cross sectional area ofdistributor channel 24 is completely filled by the sample liquid 22, thefurther transport of the sample liquid in distributor channel 24 isperformed by capillary forces which are effective within the channel.

The inflow channels 26 are arranged to branch off from distributorchannel 24 transversely to the extension thereof. Also in these inflowchannels 26, the further transport of the sample liquid 22 is performedby capillary forces. The liquid transport through the inflow channels 26will extend first to the entrance site 52 of each inflow channel 26 intothe reaction chamber 28 assigned to the channel (cf. FIG. 5). Withouttaking special measures or observing special conditions with regard tothe configuration of the inflow channels 26 and the reaction chambers28, a danger exists that the liquid front will not extend farther intothe reaction chamber 28 from the entrance site 52 of the inflow channel26.

To further guarantee a reliable liquid transport by capillary effect inthe above situation, the entrance site 52 is arranged on the upper end,facing away from the bottom wall 54 of a reaction chamber 28, of twomutually angled side faces 56 of reaction chamber 28. The overallreaction chamber 28 is of a square or at least rectangular cross section(cf. the illustration in FIGS. 1 and 5) so that corner regions 58 and60, respectively, are generated between respectively adjacent side faces56 and between the side faces 56 and the bottom face 54. By formingthese corner regions with a sufficiently small radius of curvature, aliquid meniscus can be generated in the transition region of the facesforming the respective corner regions, which meniscus—due to thetendency of the liquid to wet the adjacent regions of the faces—will bemoved along the corner regions 58,60 under the effect of capillaryforces.

Thus, the corner region 58 having the entrance region 52 of the inflowchannel 26 arranged therein, acts as an inflow groove 62. This inflowgroove 62 allows a flow of the sample liquid 22 from the inflow channel26 into reaction chamber 28. This liquid first flows along the inflowgroove 62 in the direction towards the bottom face 54 of reactionchamber 28, and flow from there along the corner regions 58 which extendcontinuously in the shape of a square, until the whole bottom ofreaction chamber 28 is wetted. In this manner, the reaction chamber 28is increasingly filled with sample liquid exclusively by use ofcapillary forces.

The filling of the plurality of reaction chambers 28 should be performedin a uniform manner and particularly simultaneously. A too suddenfilling of the reaction chambers 28 with sample liquid 22 can lead toundesirable effects because the sample liquid 22 might possibly flow offagain undesirably via the connecting channels 30 provided for venting.Therefore, it is of advantage to have the sample liquid 22 admitted intothe reaction chambers 28 in throttled fashion. For this reason, thecross sections of the inflow channels 26 are smaller than the crosssection of the distributor channel 24. The inflow channels thus form akind of throttle with increased flow resistance. This throttle effectoffers the additional advantage that, although the individual inflowchannels branch off from the distributor channel 24 at differentdistances from the sample receiving chamber 20, all of the reactionchambers 28 are filled simultaneously (with a certain delay beingtolerated).

As can be seen particularly in FIGS. 1 and 5, the inflow channels 28,when viewed along the extension of distributor channel 24, are arrangedto branch off therefrom in a mutually staggered relationship. This hasthe advantage that the liquid front advancing through the distributorchannel 24 is respectively “disturbed”—in the region where the inflowchannels 26 branch off—only by the entrance opening of an inflow channel26. Notably, if the inflow channels 26, arranged in pairs on both sidesof distributor channel 24, were to branch off opposite to each other,the liquid transport could be disturbed to an extent which would causeit to stop. In this regard, it is to be considered that an unevenness ofthe surface can sometimes massively impair the effective capillaryforces. The branching of an inflow channel 26 from the distributorchannel 24 acts like a widening of the channel which, if too large,could bring the flow to a standstill. Notably, the transport through abranched-off inflow channel 26 by capillary forces acting therein willoccur only when the liquid in distributor channel 24 covers the crosssection of the branched-off inflow channel 26. For this reason, theinflow channels 26 have cross sections small enough that they willultimately pose no obstacle to the tendency of the liquid to wet theinner walls of distributor channel 24 in spite of the branched-offinflow channel 26.

During the filling of the reaction chambers 28 with the sample liquid22, air or gas existing within these chambers is discharged via theconnecting channels 30. Each connecting channel 30 is arranged to enterthe respective reaction chambers 28 via an antechamber space 64 (cf.also FIG. 7). Antechamber space 64 is arranged on the upper end ofreaction chamber 28 and delimited in upward direction by cover film 16.The bottom wall 66 of antechamber space 64 opposite cover film 16extends obliquely downwards in the direction of reaction chamber 28. Theconfiguration of antechamber space 64 is selected such that all of theair or gas in reaction chamber 28 will be discharged when the latter isbeing filled so that, finally, the liquid level within reaction chambers28 will reach up to cover film 16 and will not be disturbed by gasbubbles and the like. As is evident particularly from FIG. 5, theconnecting channels 30 serving for the venting of the reaction chambers28 are arranged to enter the venting collecting channel 32 via widenedportions 68 which are heart-shaped when seen in plan view. Each widenedportion 68 comprises chamber portions 72 extending on both sides of theentrance 70 of connecting channel 30 and reaching to a region—relativeto the gas flow direction—upstream of the entrance site 70 and taperingtowards the venting collecting channel 32. The entrance side 70 islocated in a side face region 74 of the widened portion 68, with theside face region 74 having no corner regions arranged therein, neitherlaterally of nor below the entrance site 70. The only corner regionexisting is generated laterally of the entrance site 70 and adjacent tofilm 16. Thus, the connecting channel 30 ends within the widened portion68 in such a manner that its entrance site 70 is surrounded by arealportions. An entrance site 70 of this type has the advantage that theoncoming liquid front is stopped at the entrance site 70 because afurther transport thereof is prevented by capillary forces. This liquidfront will move on through the connecting channels 30 since, after thecomplete filling of the reaction chambers 28, the sample liquid willmove, via antechamber space 64, into the connecting channels 30 actingagain as capillaries. Thus, the widened portion 38 prevents that thesample liquid proceeds into the venting collecting channel 32.

As mentioned already above, each venting collecting channel 32 extendsfrom a reagent liquid receiving chamber 38. Contained in these receivingchambers 38 is an additional reagent liquid which is required toinitiate reactions of the sample liquid in the reaction chambers 28. Thereaction chambers 28 are advantageously provided beforehand with reagentsubstances which have been preconditioned and introduced into thereaction chambers 28 according to the examinations to be performed.Until the inflow of the sample liquid 22, these reactive substances arearranged in dried form within the reaction chambers 28.

When the reaction of the sample liquid with the reactive substancesalready contained in the reaction chambers 28 has been completed, it maybe required to induce an additional reaction. For this purpose, theconduit system which comprises the venting collecting conduits 32 andthe connecting conduits 30 as well as the widened portions 68 and up tothen has been used as a venting system, is thereafter utilized forintroducing additional reagents into the reaction chambers 28. For thisuse, it should be safeguarded that the widened portions 69 can be passedby the reagent liquid. This can be realized, for instance, byconfiguring the entrance sites 76 of the venting collecting channels 32into the widened portions 68 in such a manner that the inflow of thereagent liquid into the widened portions under the effect of capillaryforces will be guaranteed. Useful for this purpose are the samemechanisms that have been described farther above in connection with theinflow of the sample liquid 22 from the inflow channels 26 into thereaction chambers 28. By the formation of corner regions withsufficiently small rounding radii in the immediate vicinity of entrancesite 76, the inflow of reagent liquid into the chambers 72 of thewidened portions 68 through capillary forces can be obtained. As afurther alternative, it can be provided that the application of anhydraulic pressure onto the reactive liquid in the chambers 38 causesthe widened portions 68 to be filled with reactive liquid. A thirdpossibility consists in a controlled introducing of a control liquidinto the widened portions 68. (The control channels and control liquidreceiving chambers required therefore are not illustrated in theFigures.) All of the variants described here have in common that thefurther transport of the reagent substances in the reagent liquid intothe reaction chambers 28 requires that the widened portions 68 be filledwith liquid. As soon as these portions 68 have been filled with liquid,this liquid will at the entrance site 70 contact the sample liquidarranged in the connecting channel 30. The further transport of thereagents of the reagent liquid is then performed by diffusion. In otherwords, the widened portion 68 forms a bi-directional valve which,depending on the flow direction, is either in the closed condition or inthe open condition.

For the sake of completeness, it should be pointed out with reference toFIGS. 5 and 9, that also in this case, use is made of capillary forcesfor transport of the reagent liquid from the reagent receiving chambers38 into the venting collecting channels 32 joining the latter. Thismechanism is similar to the one described in connection with FIGS. 1 and6. According to FIG. 9, the venting collecting channel 32 is arranged tobranch off at the upper end facing away from the bottom wall 78 ofchamber 38. In this region, the entrance site 80 in the side walldelimiting region 82 of chamber 38 is rounded as shown in FIG. 5. Torealize a flow, based on capillary forces, out of chamber 38 intochannel 32, there is again required a sort of outflow groove 84 with aradius of curvature small enough to generate a liquid meniscus which,due to the tendency of the liquid to wet the groove 84, will move alongthis groove, in this case in the upward direction.

With reference to FIGS. 10 to 14, constructional possibilities of valveconfigurations will be discussed hereunder which make it possible tohave the liquid from the sample receiving chambers flow into theconnected distributor channels 24 in a controlled manner.

A first variant of such a valve 86 is shown in FIG. 10. In this valveconstruction 86, the distributor channel 24 extends through a widenedchannel portion 88 which is round in plan view and has a poroushydrophobic insert body 90 arranged therein. Due to its hydrophobicproperties, the body 90 will block the liquid transport by the widenedportion 88. When the sample liquid in receiving chamber 20 is subjectedto a pressure, the liquid is pressed into the widened portion 88 andthus into the porosities of the hydrophobic insert body 90. In theprocess, the porous body 90 has sample liquid flowing therethrough untilthe liquid reaches the region of the distributor channels 24 joining thewidened channel portion 88 and arranged behind the insert body 90 whenviewed in the flow direction. From then on, the further transport of theliquid is performed by capillary forces. Since the hydrophobic insertbody 90 on its surfaces is wetted by the sample liquid as a result ofthe pressure acting on the latter, the liquid flow through capillaryforces is maintained. Thus, in this manner, a valve function is realizedby liquid control (pressure control of the sample liquid).

FIGS. 11 and 12 show a further alternative valve configuration 86′. Theunderlying thought in this valve configuration 86′ is the one describedin connection with the widened portions 68 (cf. FIGS. 5 and 8). Thus,also in this configuration 86′, the distributor channel 24 includes aspecial widened channel portion 88′ which in plan view and sectionalview is provided in the manner shown in FIGS. 11 and 12. In the regionof the entrance 92 of the portion of distributor channel 24 coming fromsample receiving chamber 20, the widened portion 88′ comprises a planeside face 94 which only towards the cover film 14 is delimited by acorner region. The capillary forces thus possibly generated on bothsides of the entrance 92 on the underside of cover film 14 will notsuffice to suck the liquid from the distributor channel 24. Thus, theliquid front advancing from the sample chamber 20 through the joiningportion of the distributor channel 24, is brought to a stop at theentrance site 92. Only when pressure is applied onto the liquid of thesample receiving chamber 20, sample liquid enters the widened portion88′ and fills the same. The widened portion 88′ has an outlet 92arranged to enter the further extension of distributor channel 24. Assoon as the liquid pressed into the widened portion 88′ reaches theoutlet 96, the further transport of the sample liquid is again performedby capillary effect.

Finally, FIGS. 13 and 14 show a configuration of a valve 86″. Themechanisms and the configuration of this valve are nearly identical withthe valve configuration 86′. The difference between the two valvesresides in that the filling of the widened portion 88″ of valve 86″ isperformed not by the sample liquid but by a control liquid 98 which isinert to the sample liquid. The control liquid 98 is arranged in areceiving chamber 100 which via a control channel 102 is connected tothe widened portion 88′. The introducing of the control liquid 98 intothe widened portion 88″ can be performed by application of pressure ontothe control liquid 98 on the one hand, but also by maintaining a liquidflow by use of capillary forces on the other hand. In the latter case,the measures provided are of the type described above in connection withthe introducing of the sample liquid 22 into the reaction chambers 28,i.e. the entrance 104 of the control channel 102 into the widenedchannel portion 88″ is provided in a region in which, within the widenedchannel portion 88″, corner regions with sufficiently small roundingradii are formed, with a meniscus being generated and moving therealong.By application (cf. FIGS. 13 and 14) of control liquid into the chambers100, the switching of valve 86″ can be influenced automatically, as itwere (notably from the closed into the conductive state). To have thecontrol liquid 98 move from chamber 100 into control channel 102, usecan be made again of the mechanisms and measures described above inconnection with the outflow grooves of chambers 20 and 38.

As already mentioned above, the reaction chambers of the sample supportcan already be provided with reactive substances on the manufacturer'sside, which substances are stored in the reaction chambers in driedform. Because of the small volumes of the reaction chambers, only smallquantities of reactive substances are needed, which is useful for thedrying process.

The introduction of the sample liquid will be performed by the user. Ifthe cover film 16 does not extend into the regions of the upper side 14of base plate 12 wherein the sample receiving chambers 20 are located,the latter are freely accessible so that sample liquid can be introducedin conventional manner by pipeting. The same holds true if the coverfilm extends across the whole upper side and is provided with openingsarranged flush with the sample chambers (and the reagent liquidreceiving chambers 38). For improved protection against evaporation, itis of advantage if the cover film bridges the chambers 20 and 38. Insuch a case, the sample liquid can be inserted by puncture of the coverfilm. By way of alternative, the cover film in the region of chambers 20and 38 can be slitted, thus to be opened in the manner of a septum forintroducing liquid.

With regard to the mechanisms relevant for the liquid flowing in thecorner regions and along these, it should be noted here that therounding radii referred to in the instant description are provided inthe μm and sub-μm region. Further, generally, the rounding radius isadvantageously smaller than the smallest dimension of the channel joinedby the corner region.

1. A sample support comprising at least one sample receiving chamber fora sample liquid, a distributor channel for sample liquid, connected tosaid at least one sample receiving chamber, with at least one suchdistributor channel extending from said at least one sample receivingchamber, a plurality of reaction chambers each comprising a cavity whichis delimited by surfaces and is entered by an inflow channel branchedoff said at least one distributor channel, and a venting opening foreach reaction chamber, wherein each venting opening of each reactionchamber has a connecting channel extending therefrom and wherein aplurality of such connecting channels are arranged to enter respectivelyone venting collecting channel comprising a venting collecting opening,wherein each distributor channel and each inflow channel is dimensionedto have the liquid transport through the distributor and inflow channelseffected by capillary forces, and wherein, in each reaction chamber,said surfaces in the entrance region of the inflow channel which areprovided for delimiting said cavity are configured as a means forgenerating a capillary force causing the sample liquid to flow from theinflow channel into the reaction chamber exclusively by capillary force.2. The sample support according to claim 1, wherein each connectingchannel and/or each venting opening includes a means for preventing afurther flow of sample liquid effected by capillary forces.
 3. Thesample support according to claim 2, wherein said capillary-forceprevention means are arranged in the entrance regions of the connectingchannels into the venting collecting channels.
 4. The sample supportaccording to claim 2, wherein each of said capillary-force preventionmeans is provided as a widened portion of a connecting channel orventing opening, which widened portion respectively comprises a sidesurface with a connecting channel entering thereinto, and that theentrance region of the portion of the connecting channel extending fromthe reaction chamber is not delimited in the widened portion by anycorner regions or only by such a small number of corner regions withrounding radii generating a capillary force that the flow of the sampleliquid in the entrance region is prevented.
 5. The sample supportaccording to claim 4, wherein each venting collecting channel isarranged to extend from a reagent receiving chamber for receiving areagent liquid, with the flow of the reagent liquid performed via theventing collecting channels by capillary forces generated within theventing collecting channels, and that, within the entrance region ofeach venting collecting channel into the widened portions and/or withinthe entrance regions where the portions of the connecting channelsextending from the venting collecting channels enter the widenedportions, a means is arranged for generating a capillary force forfilling the widened portions.
 6. The sample support according to claim5, wherein each reagent receiving chamber comprises a bottom surface andside surfaces extending at an angular orientation thereto, and that theventing collecting channel assigned to a reagent receiving chamber isarranged to enter the reagent receiving chamber above said bottomsurface, and that a means for generating a capillary force to causereagent liquid to flow from the reagent receiving chamber into theventing collecting channel is arranged between said entrance and saidbottom surface.
 7. The sample support according to claim 6, wherein saidcapillary-force generating means is formed as an outflow groove having across-sectional area and shape suited to generate a flow of the reagentliquid by capillary force.
 8. The sample support according to claim 7,wherein said outflow groove is provided as a trough formed in a sidesurface.
 9. The sample support according to claim 7, wherein saidoutflow groove is provided as a transition region between two adjacentand mutually angled side surfaces, the transition region having arounding radius sufficiently small to generate capillary forces causinga flow of the reagent liquid.
 10. The sample support according to claim4, wherein each venting collecting channel is arranged to extend from areagent receiving chamber for receiving a reagent liquid, and that,within the entrance region of each venting collecting channel into thewidened portions and/or within the entrance regions where the portionsof the connecting channels extending from the venting collectingchannels enter the widened portions, a means is arranged for generatinga capillary force for filling the widened portions.